Invitrogen

Phospho-ATF2 (Thr71) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell treatment to ensure that the antibody binds to the antigen stated.

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Phospho-ATF2 (Thr71) Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of Phospho-ATF-2 pThr71 showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- Phospho-ATF-2 pThr71 Polyclonal Antibody (Product # 44-295G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Phospho-ATF2 (Thr71) Antibody in Western Blot (WB) — Western blot analysis was performed on whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), U-87 MG treated with 25 µg/mL anisomycin for 30 minutes (Lane 2), K562 (Lane 3), K562 treated with 25 µg/mL anisomycin for 30 minutes (Lane 4). The blots were probed with Anti-Phospho-ATF-2 pThr71 Rabbit Polyclonal Antibody (Product # 44-295G, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A ~ 54 kDa band corresponding to Phospho-ATF-2 pThr71 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Phospho-ATF2 (Thr71) Antibody — Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot using Phospho-ATF2 (Thr71) Polyclonal Antibody (Product # 44-295G), shows increased levels of phospho-ATF2 (Thr71) in U-87MG and K562 cell lines upon Anisomycin treatment. {TM}

Product Details

44-295G

Applications	Tested Dilution	Publications
Western Blot (WB)	1:1,000	-
Immunohistochemistry (Paraffin) (IHC (P))	1:20	-

Product Specifications
Species Reactivity	Human
Host/Isotype	Rabbit / IgG
Class	Polyclonal
Type	Antibody
Immunogen	Chemically synthesized phosphopeptide derived from a region of human ATF2 that contains threonine 71 View immunogen
Conjugate	Unconjugated
Form	Liquid
Purification	Antigen affinity chromatography
Storage buffer	Dulbecco's PBS, pH 7.3, with 50% glycerol, 1mg/mL BSA
Contains	0.05% sodium azide
Storage conditions	-20°C
RRID	AB_2533624

Product Specific Information

This antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non phosphorylated ATF2. The final product is generated by affinity chromatography using an ATF2-derived peptide phosphorylated at threonine 71. The peptide sequence is conserved in mouse, rat, chicken and frog. Suggested Western blot positive controls: NIH3T3 + anisomycin, or Jurkat + 10 µg/mL anisomycin for 60 minutes.

Target Information

The transcription factor ATF-2 (also called CRE-BP1) binds to both AP-1 and CRE DNA response elements and is a member of the ATF/CREB family of leucine zipper proteins. ATF-2 interacts with a variety of viral oncoproteins and cellular tumor suppressors and is a target of the SAPK/JNK and p38 MAP kinase signaling pathways. Various forms of cellular stress, including genotoxic agents, inflammatory cytokines and UV irradiation, stimulate the transcriptional activity of ATF-2. Cellular stress activates ATF-2 by phosphorylation of Thr69 and Thr71. Both SAPK and p38 MAPK have been shown to phosphorylate ATF-2 at these sites in vitro and in cells transfected with ATF-2.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

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Bioinformatics

Protein Aliases: Activating 2; Activating transcription factor 2; activating transcription factor 2 splice variant ATF2-var2; cAMP response element-binding protein CRE-BP1; cAMP responsive element binding protein 2, formerly; cAMP-dependent transcription factor ATF-2; CREB-2; Cyclic AMP-dependent transcription factor ATF-2; Cyclic AMP-responsive element-binding protein 2; HB16; histone acetyltransferase ATF2; MGC111558; OTTHUMP00000205345; OTTHUMP00000205347; OTTHUMP00000205348; OTTHUMP00000205350; OTTHUMP00000205352; OTTHUMP00000205353; OTTHUMP00000205354

Gene Aliases: ATF2; CRE-BP1; CREB-2; CREB2; CREBP1; HB16; TREB7

UniProt ID: (Human) P15336

Entrez Gene ID: (Human) 1386

Molecular Function: basic leucine zipper transcription factor